Abstract. RNA plays a key role in most aspects of gene regulation, and is increasingly viewed as a drug target. To fully exploit RNA in medicine and biology, innovative approaches are needed to probe the interactions between structured RNAs and small molecules with the goal of elucidating how binding alters cellular function. Riboswitches are natural RNA sensors located typically in the 5-leader of mRNAs where they bind cognate effectors to regulate downstream genes. We previously determined representative crystal structures of the three known preQ1 riboswitch classes revealing: new tertiary folds, novel determinants of ligand recognition, and diverse strategies to bury associated Shine-Dalgarno Sequences (SDSs). Nonetheless, a substantial gap still exists in the field's understanding of the underlying signal transduction pathways that connect effector binding to gene-regulatory conformation. To address this formidable challenge, we developed new tools to cogently relate structure to function: (i) we prepared a robust bacterial reporter in which a preQ1 riboswitch represses GFPuv expression in response to the effector preQ1 (EC50 6.9 nM); (ii) we developed SiM-KARTS (Single Molecule Kinetic Analysis of RNA Transient Structure), which detects effector-induced conformational changes by monitoring repeated association and dissociation events between the riboswitch SDS and a probe mimicking the 3-end of 16S ribosomal RNA. Our results show that SDS exposure occurs in `bursts' that diminish with preQ1; (iii) we developed 2-methylnicotinc acid imidazolide (NAI) and 3-nicotinoyl azide (NAz) to probe specific preQ1 riboswitch conformations that regulate the in cell GFPuv reporter, whose control status is visualized by fluorescence. These novel approaches form a strong basis to investigate the premise that discrete signal transduction networks link effector binding to riboswitch gene regulation. We will address this overarching goal in three dovetailed aims: (Aim 1) Identify preQ1 riboswitch mutants integral to signal transduction; (Aim 2) Quantify SDS accessibility and dynamics of riboswitch variants; and (Aim 3) Relate riboswitch biophysical data to in vivo chemical modification analysis to elucidate interaction networks operative in gene regulation. To our knowledge, no other group is using such tools and approaches to dissect effector- mediated signal-transduction pathways for an entire riboswitch family. We are a team of experts comprising four P.I.s with strong records in: RNA structural biology, effector binding, RNA mutagenesis, and biophysical studies (Wedekind, P.I.); single-molecule FRET and methods development (Walter, U. Michigan); RNA probing reagents, protocols, and analysis (Spitale, UC Irvine); and `RNAstructure' prediction using experimental restraints (Mathews, University of Rochester). We are uniquely qualified to perform this research. High-value outcomes include the unprecedented elucidation and in vivo validation of signal transduction pathways used by riboswitches to control translation. This work will also contribute broadly to our knowledge of ligand-mediated control of RNA conformation, while providing new tools to analyze RNA structure and function relationships.